Addiction to various drugs accounts for a major fraction of the public health burden in the U.S. One of the key features in drug addiction is the tendency of relapse despite long term abstinence. Therefore, an important goal of addiction-related research is to elucidate biochemical and cellular mechanisms that mediate this reward-based learning, and to determine how these mechanisms are affected by drugs. Catecholamine signaling is a central contributor to reward-based learning. Two major catecholamine receptors responsible for mediating reward-based learning are the D1 dopamine receptor (D1R) and 22 adrenergic receptor (22AR). Both receptors signal via the stimulatory heterotrimeric G proteins (Gs and Golf), meaning that their activation results in the generation of cyclic AMP (cAMP). In addition, both D1R and 22AR undergo rapid ligand-induced internalization after receptor activation. Internalization has been shown to promote recovery (re-sensitization) of 22AR signaling via cAMP, but this appears not to apply to the D1R. Using live cell imaging of FRET cAMP sensors, I have identified a role of internalization in promoting acute cAMP generation by both of D1R and 22AR, and over a time scale considerably more rapid than re-sensitization described previously. These results lead to the hypothesis that D1R and 22AR both require receptor internalization to potentiate rapid cAMP generation. The specific aims of this proposal investigate this hypothesis in an established model cell system (HEK293 cells) and in striatal medium spiny neurons that are relevant to reward-based learning. The Specific Aims are: 1. Does clathrin-mediated endocytosis promote rapid cAMP accumulation stimulated by Gs- coupled catecholamine receptors (22AR and D1R) in HEK293 cells? 2. Does Rab4 or Rab11 mediated recycling promote rapid cAMP accumulation stimulated by Gs-coupled catecholamine receptors (22AR and D1R) in HEK293 cells? 3. Do aspects of receptor trafficking that promote rapid cAMP accumulation of Gs-coupled catecholamine receptors (22AR and D1R) in HEK293 cells apply to primary cultured striatal neurons? 4. Does internalization-dependent signaling contribute to distinguishing the functional effects of natural ligands or clinically relevant drugs? PUBLIC HEALTH RELEVANCE: These studies are relevant to understanding how drugs of abuse produce robust behavioral reinforcement, thereby addressing the mechanistic basis of addiction.